RT Journal Article SR Electronic T1 Ex vivo SIM-AFM measurements reveal the spatial correlation of stiffness and molecular distributions in 3D living tissue JF bioRxiv FD Cold Spring Harbor Laboratory SP 2024.05.27.595975 DO 10.1101/2024.05.27.595975 A1 Shioka, Itsuki A1 Morita, Ritsuko A1 Yagasaki, Rei A1 Wuergezhen, Duligengaowa A1 Yamashita, Tadahiro A1 Fujiwara, Hironobu A1 Okuda, Satoru YR 2024 UL http://biorxiv.org/content/early/2024/06/01/2024.05.27.595975.abstract AB Living tissues each exhibit a distinct stiffness, which provides cells with key environmental cues that regulate their behaviors. Despite this significance, our understanding of the spatiotemporal dynamics and the biological roles of stiffness in three-dimensional tissues is currently limited due to a lack of appropriate measurement techniques. To address this issue, we propose a new method combining upright structured illumination microscopy (USIM) and atomic force microscopy (AFM) to obtain precisely coordinated stiffness maps and biomolecular fluorescence images of thick living tissue slices. Using mouse embryonic skin as a representative tissue with mechanically heterogeneous structures inside, we validate the measurement principle of USIM-AFM. Live measurement of tissue stiffness distributions revealed the highly heterogeneous mechanical nature of embryonic skin as well as the role of collagens in maintaining its integrity. Furthermore, quantitative comparisons of stiffness distributions of preserved tissue samples unveiled the distinct impacts of conventional tissue preservation techniques on the tissue stiffness pattern. This series of experiments highlights the importance of live mechanical testing of tissue-scale samples. Our USIM-AFM technique provides a new methodology to reveal the dynamic nature of tissue stiffness and its correlation with biomolecular distributions in live tissues and thus could serve as a technical basis for exploring tissue-scale mechanobiology.Competing Interest StatementThe authors have declared no competing interest.